Quantitative Western Blot Analysis of PsaA

Wild-type and mutant S. pneumoniae were grown under the same conditions as for ICP–MS. After reaching an A₆₀₀ of 0.4, cells were incubated with 0.1% sodium deoxycholate at 310 K for 60 min to induce lysis. Protein concentrations were determined and 10 μg of total protein was loaded into each lane. After electrophoretic separation by SDS–PAGE, proteins were transferred to a nitrocellulose membrane using the iBlot (Life Technologies) system. The blots were incubated with murine anti-PsaA serum (1:2,000; ref. 23 (link)), followed by anti-mouse IRDye 800 (LI-COR; 1:50,000), and were scanned using an Odyssey infrared imaging system (LI-COR). Band intensities were measured using the manufacturer’s application software and the results correspond to the mean (±s.e.m.) of two independent biological experiments.